1. Field of the Invention
This invention relates to detection and measurement of steroids in biological fluids, and, more particularly, detection and measurement of steroids by means of competitive immunoassay.
2. Discussion of the Art
Detection and measurement of steroids in biological fluids is important for a variety of reasons. For example, the amount of a particular steroid in a biological fluid can be used to assist in diagnosing the occurrence of an endocrinological disorder, to monitor the amount of hormone required in hormonal replacement therapy, or to assess fertility. Determination of the presence and amount of steroids in a biological fluid can be determined by competitive diagnostic assay. Small molecule, competitive diagnostic assays require a labeled component that can compete with the analyte for available antibody sites. Examples of the labeled component include radioactive tracers, fluorophore/hapten conjugates, and enzyme/hapten conjugates. Typically, the labeled component consists of the analyte or an analogue of the analyte coupled to a label. The labeled component is typically referred to as a conjugate.
Estradiol (1,3,5(10)-estratrien-3,17.alpha.-diol) is an analyte, the detection and measurement of which is of great importance in the area of fertility testing. Estradiol is secreted by the ovary and placenta. It is synthesized by the aromatization of androgens in the thecal and granulosa cells of the ovary and placenta. The aromatization is stimulated by follitropin (FSH). Estradiol synthesis in turn stimulates production of lutropin (LH) receptors necessary for the synthesis of androgen precursors.
Estradiol is important for female sexual differentiation during gestation, sexual development at the onset of puberty, and regulation of the menstrual cycle. The menstrual cycle is the result of a precise coordination of the functional characteristics of the central nervous system, the hypothalamus, the pituitary, the ovary, and the endometrium, which regulate the cyclic release of Gonadotropin Releasing Hormone (GnRH), LH and FSH, and ovarian steroids (estradiol and progesterone). Estradiol is involved in both the stimulation and inhibition of the release of the gonadotropins, exerting both a positive and a negative feedback. Early in the follicular phase, ovarian secretion of estradiol from the thecal and granulosa cells is modest. During the follicular phase, estradiol stimulates endometrial growth (repairing the endometrium after menses). Toward mid-cycle, LH production increases and results in the release of the ovum by the rupture of the developed follicle. After ovulation, estradiol secretion declines slightly. During the luteal phase, estradiol along with progesterone are secreted by the corpus luteum, stimulating further endometrial growth. If the ovum is not fertilized, there is a further drop in estradiol and progesterone. This drop in estradiol and progesterone initiates menses.
The measurement of estradiol is important for the evaluation of normal sexual development (menarche), causes of infertility (anovulation, amenorrhea, dysmenorrhea), and menopause. Normal estradiol levels are lowest at menses and during the early follicular phase (25-75 pg/mL). The levels rise in the late follicular phase to a peak of 200-600 pg/mL just before the LH surge initiates ovulation. As LH peaks, estradiol begins to decrease before rising again during the luteal phase (100-300 pg/mL). If conception does not take place, estradiol falls further to its lowest levels, thus initiating menses. If conception occurs, estradiol levels continue to rise, reaching levels of 1-5 ng/mL during the first trimester, 5-15 ng/mL during the second trimester, and 10-40 ng/mL during the third trimester. During menopause, estradiol levels remain low.
There are various methods for measuring estradiol levels in serum. However, many of these methods utilize radioactive elements as labels and suffer from several disadvantages. Several of these methods are described in U.S. Pat. No. 5,342,760 (column 1, line 54 through column 3, line 30) and are incorporated herein by reference. U.S. Pat. No. 5,342,760 discloses and claims a useful method for determination of estradiol by competitive immunoassay. However, the method claimed in this patent is only effective with a limited number of antibodies.
Progesterone (4-pregnen-3,20-dione) is an analyte, the detection and measurement of which is of great importance in the area of assessing the occurrence of ovulation, conception, the risk of abortion, or ectopic pregnancy.
In the mitochondria, cholesterol is first converted to pregnenolone via a cytochrome P-450 enzyme-dependent side chain cleavage followed by hydroxylation. Pregnenolone is then converted to progesterone in a reaction catalyzed by 3.beta.-hydroxysteroid dehydrogenase and isomerase enzymes (3.beta.-HSD). Progesterone is produced primarily by the corpus luteum of the ovary in normally menstruating women and to a lesser extent by the adrenal cortex. At approximately the sixth week of pregnancy, the placenta becomes the major producer of progesterone. In the circulation of blood, approximately 97-98% of the progesterone is bound to albumin or Cortisol Binding Protein. Progesterone is metabolized, primarily in the liver, to pregnanediol and its water soluble sulfate and glucuronide derivatives and excreted in the urine.
The major functions of progesterone are in the preparation of the uterus for implantation and maintaining pregnancy. During the follicular phase, progesterone levels remain low (0.2-1.5 ng/mL). Following the LH surge and ovulation, luteal cells in the ruptured follicle produce progesterone in response to LH. During the luteal phase, progesterone rises rapidly to a maximum of 10-20 ng/mL at the fifth to seventh day following ovulation. If pregnancy does not occur, progesterone levels decrease during the last four days of the menstrual cycle due to the regression of the corpus luteum.
If conception occurs, the levels of progesterone are maintained at mid-luteal levels by the corpus luteum until about the sixth week. At that time the placenta becomes the main source of progesterone and levels rise from approximately 10-50 ng/mL in the first trimester to 50-280 ng/mL in the third trimester.
Serum progesterone is a reliable indicator of either natural or induced ovulation because of its rapid rise following ovulation. Disorders of ovulation, including anovulation, are relatively frequent and are responsible for infertility in approximately 15 to 20% of patients. Progesterone levels are abnormally low in these patients during the mid-luteal phase.
Luteal phase deficiency is a reproductive disorder associated with infertility and spontaneous abortion. It is thought to occur in 10% of infertile women. It is believed that infertility and pregnancy wastage associated with this disorder are caused by inadequate maturation and development of the endometrium. The failure of the endometrium is thought to be attributable to insufficient progesterone production by the corpus luteum. Serum progesterone levels in the luteal phase are lower than normal in women with luteal phase deficiency.
Measurement of progesterone in the first ten weeks of gestation has been shown to be a reliable predictor and an effective tool for the diagnosis and treatment of patients with threatened abortion and ectopic pregnancy. Suppressed progesterone levels (10-15 ng/mL) in the presence of detectable amounts of human chroionic gonadotropin (hCG) is highly suggestive of threatened abortion or ectopic pregnancy, regardless of gestational age.
Typical physiological levels (ng/mL) are as follows.
______________________________________ Women: Normal Cycling; Follicular 0.5 Ovulatory 0.5-1.5 Luteal 4.0-20.0 Other; Prepubertal 0.2-0.5 Postmenopausal 0.5 Pregnancy 40-200 Men: Prepubertal 0.25 Adult 0.25 ______________________________________
It has been found to be difficult to obtain antibodies that have the appropriate affinity for the analyte relative to the labeled component along with the specificity to allow them to be used effectively in a competitive immunoassay for steroids, such as, for example, estradiol and progesterone. The sources of antibodies are limited in number, thereby resulting in either unavailability or excessively high cost. The use of any but a limited number of antibodies in a competitive assay results in an inadequate dose response, which results in inferior sensitivity, inferior precision, or both of the foregoing. In addition, even when antibodies that demonstrate an appropriate affinity for an analyte can be developed, many of these antibodies may demonstrate the undesirable property of high cross-reactivity to structurally-similar steroids. It would be desirable to provide a competitive immunoassay format capable of detecting levels of estradiol below 50 pg/mL and levels of progesterone below 1 ng/mL, concentrations which are clinically useful but difficult to measure.
The probability that a particular anti-steroid antibody/labeled component pair will be useful to prepare a sensitive assay for a given steroid can be assessed by knowledge of the dose response curve. The dose response curve for a steroid assay is a plot of the ratio of the rate of production of response in the presence of steroid analyte to the rate of production of response in the absence of steroid analyte as a function of the concentration of the steroid analyte. The dose response curve for a given steroid assay is unique for each antibody/labeled component combination used and is modulated by the competition between antibody analyte and labeled component. Consequently, for any particular anti-steroid antibody/labeled component/steroid analyte combination, a relatively steep dose response curve indicates that the particular combination will be more likely to provide a clinically useful assay.